Abstract: T-cell lymphomas have remained treatment-challenging, not least because of their remarkable heterogeneity. The lab of Robert Orlovsky (co-founder of Asylia) analyzed the membrane proteome of peripheral and cutaneous T-cell lymphoma and discovered that surface Heat shock protein (csHSP)-70 as being consistently overexpressed. While HSP70 is well established as an intracellular stress-response chaperone, its localization to the cell surface in cancer, is gaining increased attention. Considering the recent clinical successes of antibody-drug conjugates and the exceptionally high and selective overexpression of cell surface HSP70 in T-cell lymphoma, we summarize that csHSP70 presents as an exceptionally attractive target for antibody drug conjugates (ADC) for the treatment of this malignancy. We have generated a high affinity monoclonal antibody (clone 239-87) specifically recognizing cell-surface HSP70. In preliminary data, we have generated an ADC of mAb 239-87 with MMAE, which specifically bound csHSP70 on T-cell NHL cell lines but did not bind normal peripheral blood-derived cells. Linked to MMAE, the 239-87-MMAE ADC showed cytotoxic activity against both peripheral and cutaneous T-cell lymphoma lines cell lines with comparable, or greater potency than the leading clinical ADC, brentuximab vedotin. We now propose a series of critical experiments to accelerate the clinical translation of 239-87 ADCs for the treatment of T-cell lymphoma. In aim 1, we will humanize mAb 239-87 and optimize the drug-like characteristics of the current murine clone. We will study binding of the leads to soluble HSP70, and to csHSP70 in wild-type and genome edited cell models to verify that they retain csHSP70 affinity and specificity. In specific aim 2, we will convert clone 239-87 mAb into an ADC. We will first synthesize a panel of ADCs based on the optimal lead antibody molecule linked to MMAE, a maytansine, a camptothecin, and a pyrrolobenzodiazepine with cathepsin- and acid-cleavable linkers and compare their efficacy against T-cell lymphoma cell lines. Next, we will genetically modulate csHSP70 and perform internalization and quantitative flow studies to determine the minimum csHSP70 expression needed to see anti-cancer efficacy. Finally, we will verify the potential for synergy between our ADCs and agents that enhance csHSP70, such as histione deacetylase inhibitors. In Aim 3, we will validate the activity of clone 239-87 ADCs using in vivo tumor cell line-derived models. Next, we will validate the activity of selected best performers in more physiologically relevant patient-derived xenograft (PDX) models. Finally, we will perform pharmacokinetic studies in these PDXs to begin to understand potential drug distribution and toxicity parameters. Taken together, these studies will provide a proof-of-concept that HSP70-targeted ADCs have the necessary efficacy and pharmacologic properties for development as attractive drug candidates and allow us to move ...